Method of Identifying Treatment Responsive Non-Small Cell Lung Cancer Using Anaplastic Lymphoma Kinase (ALK) as a Marker

ABSTRACT

Disclosed herein are methods for identifying a subject as having NSCLC that is predicted or is likely to respond to treatment with an ALK inhibitor, for example crizotinib. The methods include identifying a sample including NSCLC tumor cells as ALK-positive or ALK-negative using immunohistochemistry (IBC) and scoring methods disclosed herein. A subject is identified as having NSCLC likely to respond to treatment with an ALK inhibitor if the sample is identified as ALK-positive and is identified as having NSCLC not likely to respond to treatment with an ALK inhibitor if the sample is identified as ALK-negative. According to certain embodiments of the methods, subjects predicted to respond to an ALK inhibitor may then be treated with an ALK inhibitor such as crizotinib.

CROSS REFERENCE TO RELATED APPLICATION

This claims the benefit of U.S. Provisional Application No. 61/704,960,filed Sep. 24, 2012, which is incorporated herein by reference in itsentirety.

FIELD

This application relates to embodiments of a method for prognosingnon-small cell lung carcinoma, particularly a method of determiningwhether a non-small lung carcinoma is predicted to respond to ananaplastic lymphoma kinase inhibitor.

BACKGROUND

Anaplastic lymphoma kinase (ALK) protein is a member of the insulinreceptor superfamily of receptor tyrosine kinases. ALK is a type Imembrane glycoprotein that is normally expressed only in the nervoussystem. An inversion within chromosome 2p, resulting in the formation ofa fusion gene product comprising portions of the echinoderm microtubuleassociated protein-like 4 (EML4) gene and the ALK gene, was discoveredin non-small cell lung carcinoma (NSCLC) cell lines and archivedclinical specimens (Soda et al., Nature 448:561-566, 2007). ALK is nowrecognized as a critical player in NSCLC, and although EML4 is thepredominant fusion partner, other fusion partner genes have beenidentified. The incidence of ALK gene rearrangements appears to rangefrom 2-7%, translating to about 6,000 ALK positive patients/year in theUnited States and about 40,000 patients/year worldwide. Importantly, ALKgene rearrangements are rarely coincident with EGFR, HER2, or KRASmutations, demonstrating that ALK positivity is a distinct diseasesubtype.

Crizotinib (XALKORI, Pfizer) is a potent receptor tyrosine kinaseinhibitor that inhibits ALK. In two clinical trials, ALK-positivelocally advanced or metastatic NSCLC patients who were treated withcrizotinib exhibited overall response rates of 50% (N=136; 95% CI: 42%,59%) and 61% (N=119; 95% CI: 52%, 70%), respectively. Thus,determination of ALK status in NSCLC patients is critical for directingpatient care. However, there remains a need for a specific, sensitive,and standardized assay for ALK status to quickly and accurately identifyNSCLC patients most likely to be responsive to crizotinib treatment.

SUMMARY

Disclosed herein are embodiments of a method for identifying a subjectas having NSCLC that is predicted to respond to treatment with an ALKinhibitor, for example crizotinib. The embodiments include identifying asample including NSCLC tumor cells as ALK-positive or ALK-negative usingimmunohistochemistry (IHC) and scoring methods disclosed herein. Asubject is identified as having NSCLC likely to respond to treatmentwith an ALK inhibitor if the sample is identified as ALK-positive and isidentified as having NSCLC not likely to respond to treatment with anALK inhibitor if the sample is identified as ALK-negative. In someexamples, a sample is labeled with an anti-ALK antibody and the sampleis identified as ALK-positive if at least one tumor cell with stronggranular cytoplasmic staining is present in the sample.

Some embodiments of the disclosed method also include selecting asubject having an ALK-positive NSCLC tumor for treatment with an ALKinhibitor. Additional embodiments of the disclosed method also includetreating a subject identified as having NSCLC that is predicted toresponse to treatment, such as by administering an ALK inhibitor (suchas crizotinib) to a subject having an ALK-positive NSCLC tumor alone orin combination with other known or future developed treatments.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are digital images of two exemplary ALK-positive casesshowing homogeneous ALK IHC expression on anti-ALK (D5F3) staining (leftpanels) and matched rabbit monoclonal negative control Ig staining(right panels).

FIGS. 2A-C are digital images of three exemplary ALK-positive casesshowing heterogeneous ALK IHC expression on anti-ALK (D5F3) staining(left panels) and matched rabbit monoclonal negative control Ig staining(right panels). Strong cytoplasmic staining is present in the anti-ALKstained samples, but the intensity varies.

FIG. 3 is a digital image showing punctate granular staining inglandular epithelial cells in a sample stained with anti-ALK (D5F3)antibody (left panel). Less evident staining of glandular epithelialcells was seen in the matched rabbit monoclonal negative control Igslide (right panel). The glandular epithelial staining was present innormal tissue elements, but not cancer cells and was excluded in slideinterpretation.

FIG. 4A is a digital image showing staining of apparent neuronal tissueelements in a sample stained with anti-ALK (D5F3) antibody (left panel)and the lack of staining in the matched rabbit monoclonal negativecontrol Ig slide (right panel). FIG. 4B is a digital image showingstaining of neural tissue in a sample stained with anti-ALK (D5F3)antibody (left panel) and in the matched rabbit monoclonal negativecontrol Ig slide (right panel). The staining was excluded in slideinterpretation because it was present in normal tissue elements orcells.

FIG. 5 is a digital image showing granular stippling present in alveolarmacrophages in a sample stained with anti-ALK (D5F3) antibody (leftpanel) and the lack of staining in the matched rabbit monoclonalnegative control Ig slide (right panel). This staining was excluded inslide interpretation because it was present in normal cells, not tumorcells.

FIG. 6A is a digital image showing staining of mucin in a sample stainedwith anti-ALK (D5F3) antibody (left panel) and in the matched rabbitmonoclonal negative control Ig slide (right panel). There was nostaining in the normal tissue and tumor cells, therefore this sample wasinterpreted to be ALK-negative. FIG. 6B is a digital image showingstaining of mucin in a sample stained with anti-ALK (D5F3) antibody(left panel) and in the matched rabbit monoclonal negative control Igslide (right panel). This sample was interpreted to be ALK-positivebecause the normal tissue was negative but the tumor cells were stained.

FIGS. 7A and B are digital images showing staining of some lymphocytesin a sample stained with anti-ALK (D5F3) antibody (left panels) and lessevident (FIG. 7A) or negative (FIG. 7B) staining in the matched rabbitmonoclonal negative control Ig slide (right panels). This staining wasexcluded in slide interpretation because it was present in normal cells.

FIGS. 8A and B are digital images of two exemplary ALK-negative casesshowing absence of ALK IHC expression in tumor cells on anti-ALK (D5F3)staining (left panels) and matched rabbit monoclonal negative control Igstaining (right panels).

FIGS. 9A and B are digital images of exemplary ALK-negative casesshowing weak granular cytoplasmic staining on both anti-ALK (D5F3)staining (left panels) and matched rabbit monoclonal negative control Igstaining (right panels).

FIGS. 10A-D are digital images of exemplary ALK-negative cases showingcytoplasmic staining on anti-ALK (D5F3) staining (left panels), which ismore notable than on the matched rabbit monoclonal negative control Igstaining (right panels). The cases were interpreted as ALK-negative dueto the lack of strong cytoplasmic staining.

FIG. 11 is a digital image of membrane/cytoplasmic staining in a samplestained with anti-ALK (D5F3) antibody (left panel) and the matchedrabbit monoclonal negative control Ig slide (right panel). Althoughstaining was more prevalent on the slide stained with anti-ALK antibodythan the negative control, the result was interpreted as ALK-negativebecause it was not strong granular cytoplasmic staining.

DETAILED DESCRIPTION I. Abbreviations

-   -   ALK anaplastic lymphoma kinase    -   DAB 3,3′-diaminobenzidine tetrahydrochloride    -   FISH fluorescent in situ hybridization    -   H&E hematoxylin & eosin    -   HQ 3-hydroxyquinoxaline-2-carboxylic acid    -   HRP horseradish peroxidase    -   IHC immunohistochemistry    -   NSCLC non-small cell lung carcinoma

II. Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising anantibody” includes single or plural antibodies and is consideredequivalent to the phrase “comprising at least one antibody.” The term“or” refers to a single element of stated alternative elements or acombination of two or more elements, unless the context clearlyindicates otherwise. As used herein, “comprises” means “includes.” Thus,“comprising A or B,” means “including A, B, or A and B,” withoutexcluding additional elements. Dates of GenBank Accession Nos. referredto herein are the sequences available at least as early as Sep. 24,2012.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Anaplastic lymphoma kinase (ALK): A member of the insulin receptorsuperfamily of receptor tyrosine kinases. ALK comprises an extracellulardomain, a single pass transmembrane domain and an intracellular tyrosinekinase domain. Chromosomal rearrangements resulting in ALK fusion genesare found in several tumor types, including anaplastic large celllymphoma, neuroblastoma, and non-small cell lung cancer. ALK/EML4 is themost common ALK gene fusion; additional ALK fusions include ALK/RANBP2,ALK/ATIC, ALK/TFG, ALK/NPM1, ALK/SQSTM1, ALK/KIF5B, ALK/CLTC, ALK/TPM4,and ALK/MSN.

ALK sequences are publically available, for example from GenBank®sequence database (e.g., accession numbers NP_(—)004295 (protein), andNM_(—)004304 (nucleic acid)). One of ordinary skill in the art canidentify additional ALK nucleic acid and protein sequences, includingALK variants and/or ALK gene fusions.

An ALK inhibitor is a molecule that inhibits or decreases ALK activity,such as ALK tyrosine kinase activity. In some examples, an ALK inhibitorcan be a small molecule, a protein (such as an antibody), or a nucleicacid (such as an antisense molecule). An ALK inhibitor may inhibit ordecrease binding of a ligand (such as pleiotrophin) to ALK and thusdecrease ALK tyrosine kinase activity. An ALK inhibitor may alsodirectly inhibit or decrease ALK tyrosine kinase activity, for example,an ATP-competitive inhibitor (such as crizotinib). Molecules thatdecrease or inhibit expression of ALK, such as antisense molecules, arealso ALK inhibitors. In some examples, ALK inhibitors inhibit ordecrease activity of a genetically altered ALK, such as an ALK genefusion (including, but not limited to ALK/EML4 gene fusions). The ALKinhibitor may specifically inhibit ALK tyrosine kinase activity or mayinhibit other receptor tyrosine kinase activity (such as c-Met/HGFRactivity), in addition to inhibiting ALK tyrosine kinase activity.Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, that is, molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen(such as ALK). Exemplary antibodies include monoclonal, polyclonal, andhumanized antibodies.

A naturally occurring antibody (such as IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. As used herein, the term antibodyalso includes recombinant antibodies produced by expression of a nucleicacid that encodes one or more antibody chains in a cell (for example seeU.S. Pat. No. 4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP256,654; EP 120,694; EP 125,023; Faoulkner et al., Nature 298:286, 1982;Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol.2:239, 1984).

The term antibody also includes an antigen binding fragment of anaturally occurring or recombinant antibody. Specific, non-limitingexamples of binding fragments encompassed within the term antibodyinclude Fab, (Fab′)₂, Fv, and single-chain Fv (scFv). Fab is thefragment that contains a monovalent antigen-binding fragment of anantibody molecule produced by digestion of whole antibody with theenzyme papain to yield an intact light chain and a portion of one heavychain or equivalently by genetic engineering. Fab′ is the fragment of anantibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule.(Fab′)₂ is the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction orequivalently by genetic engineering. F(Ab′)₂ is a dimer of two FAb′fragments held together by disulfide bonds. Fv is a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains.Single chain antibody (“SCA”) is a genetically engineered moleculecontaining the variable region of the light chain, the variable regionof the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Methods of making thesefragments are routine in the art.

Chromogen: A substance capable of conversion to a colored product, suchas a pigment or dye. Certain chromogens are electron donors that, whenoxidized become a colored product. Production of a colored product, andthe property of becoming insoluble upon chemical conversion, such as byoxidation, make chromogens useful for IHC. Particular examples ofchromogenic compounds, without limitation, include 3,3′-diaminobenzidine(DAB), 4-nitrophenylphospate (pNPP), Fast Red, Fast Blue,bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT),BCIP/NBT, AP Orange, AP Blue, tetramethylbenzidine (TMB),2,2′-azino-di-[3-ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine,4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG),o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside(X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), New Fuchsin, iodonitrotetrazolium (INT), tetrazoliumblue and tetrazolium violet.

Contact: To bring one agent into close proximity to another agent,thereby permitting the agents to interact. For example, an antibody canbe applied to a microscope slide or other surface containing abiological sample, thereby permitting detection of proteins in thesample that are specifically recognized by the antibody.

Crizotinib: A receptor tyrosine kinase inhibitor that potently inhibitsALK. Crizotinib (also known as PF-02341066 or XALKORI, Pfizer) is anorally available selective ATP-competitive small molecule inhibitor ofALK and c-Met/HGFR tyrosine kinases and their oncogenic variants. See,e.g., U.S. Pat. Nos. 7,230,098; 7,825,137; 7,858,643; and 8,217,057;each of which is incorporated herein by reference in its entirety.Crizotinib can be used to treat patients with ALK-positive NSCLC.

Detect: To determine if an agent is present or absent. In some examplesthis can further include quantification. For example, use of an antibodyspecific for a particular protein (e.g., ALK) permits detection of theprotein in a sample, such as a sample containing NSCLC tissue. Inparticular examples, an emission signal from a detectable label (such asan increase in the signal if the target is present) is detected.Detection can be in bulk, so that a macroscopic number of molecules canbe observed simultaneously. Detection can also include identification ofsignals from single molecules using microscopy and such techniques astotal internal reflection to reduce background noise.

Label: An agent capable of detection, for example by spectrophotometry,flow cytometry, or microscopy (such as light microscopy). For example,one or more labels can be attached to an antibody, thereby permittingdetection of the target protein. Exemplary labels include radioactiveisotopes, fluorophores, ligands, chemiluminescent agents, haptens,enzymes, and combinations thereof.

Non-small cell lung carcinoma (NSCLC): Any type of lung cancer otherthan small cell lung carcinoma. NSCLC includes squamous cell carcinoma(SQCC), adenocarcinoma (ADC), and large cell carcinoma. Both ADC andlarge cell carcinoma are classified as non-squamous cell type carcinoma.ADC can be grouped into subclasses, including acinar carcinoma,papillary carcinoma, bronchoalveolar carcinoma (BAC), solid tumor, andmixed subtypes (2004 World Health Organization classification of lungtumors, Beasley et al., Semin. Roentgenol. 40:90-97, 2004). ADC accountsfor about 40% of all lung cancers and is the most common form of lungcancer among individuals who have never smoked. ADC is classified as anon-squamous cell type of NSCLC. Histologically, ADC shows glandformation, papillary structures, or solid growth with mucin production.Large cell carcinoma includes the subclasses giant cell tumors, clearcell carcinoma, adenosquamous carcinoma, and undifferentiated carcinoma.

Normal cells or tissue: Non-tumor, non-malignant cells and tissue.

Sample: A biological specimen containing genomic DNA, RNA (includingmRNA), protein, or combinations thereof, obtained from a subject.Examples include a specimen containing at least one NSCLC cell (an“NSCLC sample”), for example, a tissue or tumor biopsy, fine needleaspirate, bronchoalveolar lavage, pleural fluid, sputum, surgicalspecimen, lymph node, an NSCLC metastasis, peripheral blood, or autopsymaterial. In other examples, a sample includes a control sample, such asa non-NSCLC cell or tissue sample.

Sensitivity and specificity: Statistical measurements of the performanceof a binary classification test. Sensitivity measures the proportion ofactual positives which are correctly identified (e.g., the percentage ofNSCLC tumors that are identified as being ALK-positive that areidentified as ALK-positive by another method, such as FISH). Specificitymeasures the proportion of negatives which are correctly identified(e.g., the percentage of NSCLC tumors identified as ALK-negative thatare identified as ALK-negative by another method, such as FISH).

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals, such as veterinary subjects. In aparticular example, a subject is one who has or is suspected of havinglung cancer, such as NSCLC.

Therapeutically effective amount: A dose sufficient to preventadvancement, delay progression, or to cause regression of a disease, orwhich is capable of reducing symptoms caused by the disease, such ascancer, for example lung carcinoma (such as NSCLC).

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity. An example includescontacting an antibody with a NSCLC sample sufficient to allow detectionof one or more target molecules (e.g., ALK) in the sample.

III. Methods of Identifying a Subject as Having a Tumor Likely toRespond to ALK Inhibitors

Disclosed herein are embodiments of a method for identifying a subjectas having NSCLC that is predicted to respond to treatment with an ALKinhibitor, for example crizotinib. The embodiments include identifyingan NSCLC tumor as ALK-positive or ALK-negative usingimmunohistochemistry (IHC) and scoring methods disclosed herein. Asubject is identified as having NSCLC likely or predicted to respond totreatment with an ALK inhibitor if the NSCLC tumor is identified asbeing ALK-positive and is identified as having an NSCLC tumor not likelyor not predicted to respond to treatment with an ALK inhibitor if theNSCLC tumor is identified as ALK-negative.

Current methods for selecting a subject with NSCLC for treatment with anALK inhibitor are based on determining whether the subject's tumor isALK-positive utilizing a fluorescent in situ hybridization (FISH) assay.However, FISH presents several disadvantages compared to the methodsdisclosed herein. FISH assays are more time consuming, more expensive,and require substantially more specialized expertise for interpretationof results than IHC methods, such as those disclosed herein. Inaddition, the methods disclosed herein provide a “binary” scoring methodthat identifies NSCLC tumors as ALK-positive or ALK-negative with highsensitivity and specificity.

Rapid and accurate identification of ALK-positive NSCLC (e.g., having anALK gene rearrangement and/or expressing ALK in the tumor) is criticalfor guiding patient care. Subjects with ALK-positive NSCLC treated withan ALK inhibitor (crizotinib) exhibited overall response rates ofgreater than 50% (e.g., Kwak et al., N. Engl. J. Med. 363:1693-1703,2010; Shaw et al., Lancet Oncol. 12:1004-1012, 2011). ALK generearrangement in NSCLC is estimated to have a prevalence of about 2-7%.Therefore, accurate identification of ALK-positive NSCLC is needed toselect subjects most likely to benefit from ALK inhibitor therapy.However, the currently used FISH “break-apart” assay presentssignificant challenges in sample interpretation. For example,intra-chromosomal gene rearrangements may produce subtle, difficult todetect, signal splitting, which leads to potential false negativeresults. In addition, the FISH assay requires enumeration of signalpattern for 50 nuclei. If less than 5 cells out of 50 are positive, thesample is considered to be ALK-negative; if more than 25 cells out of 50are positive the sample is considered to be ALK-positive. However, if5-25 positive cells are present, the sample is equivocal and a secondreader is required; then, if the average percent positive cells is >15%,the sample is considered to be ALK-positive. Thus, the FISH assay canproduce a significant number of “equivocal” results, necessitating timeconsuming and costly additional testing. Furthermore, samples are nottested by FISH if they do not meet the minimum tumor content requirementof at least 50 cells, so in many cases specimens such as fine needleaspirates or FFPE cytology samples are never tested by FISH. Finally,FISH has a failure rate of about 10-30%, meaning that many specimenswill never have an ALK result and ALK-positive subjects may be missedand not obtain optimal treatment.

In contrast, the disclosed embodiments utilize IHC detection of ALK. IHCmethods are rapid, routine in anatomical pathology laboratories, utilizelight microscopy (as compared to FISH, which requires specializeddark-field fluorescence microscopy), are less expensive than FISH, anddo not require specialized expertise for interpretation. In addition,the disclosed embodiments can be automated, which provides standardizedstaining and further improves sensitivity and specificity of the assay.Another advantage of the disclosed IHC methods is that there is nopre-specified tumor cell content requirement (whereas the FISH assayrequires at least 50 tumor cells). In addition, the disclosed IHC methodenables the pathologist or reader to evaluate the entire tissue sample(in contrast to FISH methods). For FISH, only a portion of the sampleidentified as containing tumor is contacted with the FISH reagents.Therefore, if ALK is present in a different tumor area on the tissue, itwill not be identified. In contrast, the disclosed method enables thereader to see the entire tumor on the slide, in the context of theentire tissue sample. Finally, the scoring methods disclosed herein are“binary” (positive or negative) and therefore provide unequivocalresults. Even previous IHC methods for detecting ALK-positive NSCLCtumors produced frequent equivocal results requiring additional testing(e.g., Yi et al., J. Thorac. Oncol. 6:459-465, 2011) or utilizedsemiquantitative grading of staining intensity and estimation ofpercentage of immunoreactive tumor cells (e.g., Mino-Kenudson et al.,Clin. Cancer Res. 16:1561-1571, 2010). However, the disclosed IHC andscoring methods provide sensitive and specific identification of anNSCLC tumor as ALK-positive even if only a few tumor cells (or even onlyone tumor cell) exhibit strong granular cytoplasmic staining with an ALKantibody.

A. Detection of ALK

In particular examples, a sample obtained from the subject is analyzedto determine if it contains ALK protein, such as detectable levels ofALK protein in one or more tumor cells. Thus, the sample can be analyzedto detect or measure the presence of ALK protein in the sample, forexample a qualitative or semi-quantitative measurement. In particularexamples, the disclosed methods utilize qualitative measurement of thepresence of ALK protein in tumor cells in the sample.

The disclosed embodiments utilize IHC to detect ALK protein in a samplefrom the subject. IHC is a method of determining the presence ordistribution of an antigen (such as a protein) in a sample (such as anNSCLC sample, for example, a portion or section of tissue includingNSCLC tumor cells or tissue) by detecting interaction of the antigenwith a specific binding agent, such as an antibody. A sample includingan antigen (such as a target antigen) is incubated with an antibodyunder conditions permitting antibody-antigen binding. Antibody-antigenbinding can be detected by means of a detectable label conjugated to theantibody (direct detection) or by means of a detectable label conjugatedto a secondary antibody, which is raised against the primary antibody(e.g., indirect detection). In other examples of indirect detection,antibody-antigen binding is detected by means of a detectable labelconjugated to a tertiary antibody which is capable of binding to asecondary antibody (e.g., is raised against the secondary antibody or israised against a molecule conjugated to the secondary antibody, such asa hapten). Exemplary detectable labels that can be used for IHC include,but are not limited to, radioactive isotopes, fluorochromes (such asfluorescein, fluorescein isothiocyanate, and rhodamine), haptens,enzymes (such as horseradish peroxidase or alkaline phosphatase), andchromogens (such as 3,3′-diaminobenzidine (DAB) or Fast Red). In someexamples, detection of antigen-antibody binding also includes signalamplification (such as tyramide signal amplification or relatedmethods). The signal amplification method may include methods describedin U.S. Pat. Publ. No. 2012/0171668, incorporated by reference herein inits entirety.

In some examples, the specific binding agent is an antibody, such as apolyclonal or monoclonal antibody, or fragment thereof. In someexamples, the antibody is a humanized antibody. In some examples, theantibody is a chimeric antibody. If desired, the antibody can include adetectable label to permit detection and in some cases quantification ofthe target protein/antibody complex. In other examples, the antibody isdetected with an appropriate labeled secondary antibody. In additionalexamples, the antibody is detected with an appropriate labeled tertiaryantibody.

In some examples, the antibody for ALK is obtained from Ventana MedicalSystems, Inc. (Tucson, Ariz.). In specific examples, the antibody isanti-ALK D5F3 rabbit monoclonal antibody (Ventana, Catalog No.790-4794). However, a person of ordinary skill in the art willappreciate that other antibodies that can be used in the methodsprovided herein are commercially available from other sources,including, but not limited to anti-ALK 5A4 antibody (Abcam, Cambridge,Mass.; Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-ALK ALK1antibody (Dako, Carpinteria, Calif.), and anti-ALK D9E antibody (CellSignaling Technology, Danvers, Mass.). One of ordinary skill in the artcan select additional anti-ALK antibodies, including those now availableor developed in the future, that can be used in the disclosed methods.

In some examples, a lung cancer sample (such as a sample including NSCLCtumor tissue) is obtained, and processed for IHC. For example, thesample can be fixed and embedded, for example with formalin andparaffin. The sample can then be mounted on a support, such as a glassmicroscope slide. For example, the sample can be sliced into a series ofthin sections (for example, using a microtome), and the sections mountedonto a microscope slide. In some examples, a single slide includesmultiple tissue sections from the same lung cancer sample or sectionsfrom the same lung cancer sample can be placed on different slides.Different sections of the lung cancer (e.g., NSCLC tumor) sample canthen be individually labeled with different antibodies, for example ananti-ALK antibody and a negative control antibody (for example, anantibody that does not specifically bind to an endogenous antigen in thesample). That is, one section can be labeled with anti-ALK antibody andanother section can be labeled with a negative control antibody (such asan antibody that binds to a target that does not occur endogenously inthe sample). In some examples, the slide labeled with an anti-ALKantibody (or a negative control antibody) is also stained withhematoxylin and eosin (H&E) (for example, to provide morphological orhistological information, such as to discriminate cell types in thesample). In other examples, a separate slide from the same subject islabeled with H&E (such as a serial section from the same NSCLC sample).In some examples, additional proteins of interest can be detected in thesame or additional tissue samples by labeling with further antibodies(for example, anti-EGFR antibodies, anti-RAS antibodies, and/oranti-HER2 antibodies). In some examples, an automated slide stainer(such as BENCHMARK instruments from Ventana Medical Systems, for exampleBENCHMARK XT or BENCHMARK GX instruments) can be used to stain andprocess the slides.

In some examples, detecting ALK protein in the sample includes indirectdetection of binding of the anti-ALK antibody to the sample (forexample, the anti-ALK (primary) antibody is not detectably labeled). Forexample, the sample is contacted with the anti-ALK antibody (such asanti-ALK D5F3 antibody) under conditions sufficient for the anti-ALKantibody to bind to ALK protein in the sample. The sample is thencontacted with a secondary antibody that can specifically bind to theanti-ALK antibody (such as an anti-rabbit antibody, if the anti-ALKantibody is a rabbit antibody) under conditions sufficient for thesecondary antibody to bind to the anti-ALK antibody. The secondaryantibody can be detectably labeled. The detectable label can beconjugated to the secondary antibody. In some examples, the detectablelabel conjugated to the secondary antibody can be directly detected(such as a fluorescent label, or an enzyme, which can produce adetectable reaction product in the presence of suitable substrate). Inother examples, the secondary antibody is conjugated to one or morehaptens (such as fluorescein, dinitrophenyl, biotin, or3-hydroxyquinoxaline-2-carboxylic acid (HQ)). The sample is thencontacted with a tertiary antibody that can specifically bind thehapten-conjugated secondary antibody (for example, an anti-haptenantibody, such as an anti-HQ antibody) under conditions sufficient forthe tertiary antibody to bind to the hapten. In some examples, thetertiary antibody is conjugated to a detectable label, such as an enzyme(for example, horseradish peroxidase (HRP) or alkaline phosphatase(AP)). The sample is then contacted with one or more reagents thatproduce a detectable reaction product in the presence of the enzyme. Insome examples, the sample is contacted with an HRP substrate (such ashydrogen peroxide) and a chromogen (such as DAB) that produces avisually detectable product in the presence of HRP. In some examples,detecting ALK protein in the sample is carried out using OPTIVIEW DABIHC Detection Kit (Ventana Medical Systems, Inc., Tucson, Ariz., CatalogNo. 760-700).

In further examples, detecting ALK protein in the sample includesindirect detection including signal amplification. In some examples,signal amplification allows unequivocal detection of ALK positivespecimens which may exhibit only weak staining without signalamplification. Signal amplification methods for IHC are known to one ofordinary skill in the art. In some examples, signal amplificationincludes CAtalyzed Reporter Deposition (CARD), also known as TyramideSignal Amplification (TSA™). In one variation of this method anenzyme-conjugated secondary antibody (such as an HRP-conjugatedsecondary antibody) binds to the primary antibody. Next a substrate ofbiotinylated tyramide (tyramine is 4-(2-aminoethyl)phenol) is used,which presumably becomes a free radical when interacting with the HRPenzyme. The phenolic radical then reacts quickly with the surroundingmaterial, thus depositing or fixing biotin in the vicinity. This processis repeated by providing more substrate (biotinylated tyramide) andbuilding up more localized biotin. Finally, the “amplified” biotindeposit is detected with streptavidin attached to a fluorescentmolecule. Alternatively, the amplified biotin deposit can be detectedwith avidin-peroxidase complex, that is then contacted with DAB toproduce a brown color.

In other examples, signal amplification includes contacting the samplewith hydrogen peroxide and a tyramide-HQ conjugate after contacting thesample with an HRP-conjugated tertiary antibody under conditionssufficient for depositing HQ at or near the site of the primary antibodybound to the sample. The sample is then contacted with anenzyme-conjugated antibody (such as an HRP- or AP-conjugated antibody)that specifically binds to HQ. In some examples, this enzyme-conjugatedantibody is the same as the HRP-conjugated tertiary antibody. In otherexamples, the enzyme-conjugated antibody is a different antibody thanthe HRP-conjugated tertiary antibody. The sample is then contacted withone or more reagents that produce a detectable reaction product in thepresence of the enzyme. In some examples, the sample is contacted withan HRP substrate (such as hydrogen peroxide) and a chromogen (such asDAB) that produces a visually detectable product in the presence of HRP.In some examples, signal amplification is carried out using OPTIVIEWAmplification Kit (Ventana Medical Systems, Inc., Tucson, Ariz., CatalogNo. 760-099).

B. Scoring Samples as ALK-Positive or ALK-Negative

To score samples as ALK-positive or ALK-negative, an NSCLC sample withdetectably labeled ALK (for example, one or more slides, such as 1, 2,3, 4, or 5 slides) is used. In some examples, the NSCLC sample can belabeled with an antibody specific for ALK and appropriately labeledsecondary and/or tertiary antibodies, for example as described inSection A, above. In one non-limiting example, OPTIVIEW DAB IHCDetection Kit and OPTIVIEW Amplification Kit are used as per themanufacturer's instructions (Ventana Medical Systems, Inc., Catalog Nos.760-700 and 760-099, respectively).

The anti-ALK labeled NSCLC sample (or a digital image thereof) isvisually inspected (for example, with or without light microscopy), forexample by a pathologist. In some examples, an entire sample (such as anentire tissue section) is visually inspected, for example using lightmicroscopy, for example at about 2×-20× magnification. In otherexamples, at least one field of view (such as at least 2, 3, 4, or 5different fields of view) is visually inspected. A field of view is anarea of a sample (for example, a tissue section) to be analyzed bymicroscopy, which is smaller than the entire section or entire digitalimage of a section. In some examples, a field of view is an area of asample visible at 2×, 5×, 10×, 20×, 40×, or 60× magnification (such as2×-20× magnification).

The disclosed methods can be used to identify (for example, score) atumor as ALK-positive or ALK-negative, for example to provide prognosticinformation, such as likely responsiveness of NSCLC to an ALK inhibitor.Tumor (for example neoplastic) cells are evaluated for presence of thedetectable label (staining), indicating expression of ALK protein in thetumor cells. A sample that has presence of strong granular cytoplasmicstaining in tumor cells (any percentage of tumor cells) is identified orscored as being ALK-positive. Strong granular cytoplasmic staining in atleast one tumor cell (such as 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more tumor cells) ina sample labeled with an anti-ALK antibody is scored as an ALK-positivesample. For example, strong granular staining in about 1-100,000 cells(such as 10-100,000 cells, 1000-100,000 cells, 1000-50,000 cells,10,000-50,000 cells, 10-50,000 cells, 500-50,000 cells, or about1000-10,000 cells) in a sample labeled with an anti-ALK antibody isscored as an ALK-positive sample. In some examples, the strong granularcytoplasmic staining is distributed homogeneously throughout theneoplastic portions of the tumor. In other examples, the strong granularcytoplasmic staining is heterogeneously distributed throughout theneoplastic portions of the tumor. In either example, presence of stronggranular cytoplasmic staining in neoplastic portions of the tumor (e.g.,in at least one or more tumor cells) is scored as ALK-positive.

If strong granular cytoplasmic staining is not present in at least onetumor cell in a sample labeled with an anti-ALK antibody, the sample isscored as an ALK-negative sample. In some examples, an NSCLC sample withno cytoplasmic staining (for example, no staining above background ornegative control levels), weak cytoplasmic staining, and/or moderatecytoplasmic staining in tumor cells is scored as an ALK-negative sample,so long as no tumor cells in the sample have strong granular cytoplasmicstaining. A sample with weak cytoplasmic staining and/or moderatecytoplasmic staining in any number of tumor cells is scored asALK-negative, unless at least one tumor cell in the sample with stronggranular cytoplasmic staining is present, in which case the sample isscored as ALK-positive.

Methods of determining staining intensity (for example,semi-quantitative IHC methods) are known to one of ordinary skill in theart. In some examples, strong granular cytoplasmic staining includesstaining in one or more tumor cells that would be identified as “3+”intensity of staining by one of ordinary skill in the art (for example,utilizing a scale of 0, no staining above background, 1+ weak intensitystaining, 2+ moderate intensity staining, and 3+ strong intensitystaining). Therefore, in some examples, presence of at least one cellwith cytoplasmic staining intensity of 3+(e.g., strong staining) isconsidered to be an ALK-positive sample. In some examples, presence in asample of cytoplasmic staining that would be identified as 1+(e.g., weakstaining) or 2+(e.g., moderate staining) intensity by one of ordinaryskill in the art in any number of tumor cells is scored as anALK-negative sample, unless there is also at least one tumor cell withstrong granular cytoplasmic staining (e.g., 3+ staining intensity)present in the sample.

One of ordinary skill in the art can identify portions of the samplewhich are neoplastic (e.g., tumor cells) and portions of the samplewhich are normal tissue or cells, for example based on morphologicaland/or histological characteristics. In some examples, the sample isstained with H&E (for example the same sample that is labeled with theanti-ALK antibody or an adjacent tissue section) to assist inidentifying tissue and cell morphology.

In some examples of the disclosed methods, one or more necrotic tumorareas may be present in a labeled sample. Staining of necrotic tumorareas or necrotic tumor cells (for example, tumor areas with loss ofnuclei and inflammatory infiltrate with or without preserved cellularoutlines) with the anti-ALK antibody is not considered to be positivestaining of tumor cells and is excluded from evaluation of the samplefor strong granular cytoplasmic staining of tumor cells. Samples withany intensity staining (including strong intensity staining) of necrotictumor cells or areas are considered to be ALK-negative, unless there isalso at least one tumor cell with strong granular cytoplasmic stainingpresent in the sample.

In other examples of the disclosed methods, staining of non-tumor cellsor normal cell elements, even strong granular cytoplasmic staining, isnot considered to be positive staining and is excluded from evaluationof the sample. For example, cells of neural origin (such as nerve cellsand/or ganglion cells) are known to express ALK (e.g., Iwahara et al.,Oncogene 14:439-449, 10997). Therefore, staining of neurons or ganglia(or other cells of neural origin) is not included in the evaluation ofthe sample. Samples with any intensity staining of cells of neuralorigin are considered to be ALK-negative, unless there is also at leastone tumor cell with strong granular cytoplasmic staining present in thesample.

In additional examples, anti-ALK staining of normal mucosa or glandularepithelial cells (even strong granular cytoplasmic staining) is notconsidered to be ALK positive staining and is excluded from evaluationof the sample. Samples with any intensity staining of normal mucosa orglandular epithelial cells are considered to be ALK-negative, unlessthere is also at least one tumor cell with strong granular cytoplasmicstaining present in the sample. Similarly, anti-ALK staining of alveolarmacrophages (for example, light granular cytoplasmic stippling) orinfiltrating lymphocytes is not considered to be ALK positive stainingand is excluded from evaluation of the sample. Samples with anyintensity staining of alveolar macrophages or infiltrating lymphocytesare considered to be ALK-negative, unless there is also at least onetumor cell with strong granular cytoplasmic staining present in thesample.

In further examples, mucin is stained by the anti-ALK antibody and isnot considered to be ALK positive staining and is excluded fromevaluation of the sample. Samples with any intensity staining of mucinare considered to be ALK-negative, unless there is also at least onetumor cell with strong granular cytoplasmic staining present in thesample.

In some examples, the scoring method also includes comparing theanti-ALK labeled NSCLC sample with one or more controls labeled with theanti-ALK antibody (for example, controls assayed in the same IHC run asthe NSCLC sample). In some examples, the control includes a positivecontrol, such as a sample including cells known to be ALK-positive (forexample H228 cells). In other examples, the control includes a negativecontrol, such as a sample including cells known to be ALK-negative (forexample Calu-3 cells). In some examples, the positive and/or negativecontrol samples are system-level controls to ensure proper functioningof assay reagents and instruments. In one example, the controls includeboth a positive and a negative control (for example, ALK 2 in 1 ControlSlides, Ventana Medical Systems, Inc., Catalog No. 781-4796).

In other examples, the negative control includes an NSCLC sample stainedwith a negative control antibody. In some examples, the negative controlantibody is an antibody that binds specifically to a target antigen thatis not endogenously present in an NSCLC sample. In some examples, thenegative control antibody is an immunoglobulin, such as a monoclonalantibody. Staining with the negative control antibody can be used toevaluate the level of background staining in a sample from the subject.In some examples, the NSCLC sample stained with the negative controlantibody is an NSCLC sample from the same subject (such as an adjacentor serial section from the sample) as the sample stained with theanti-ALK antibody. In other examples, the NSCLC sample stained with thenegative control antibody is from a different subject than the samplestained with the anti-ALK antibody.

C. Samples

Methods of obtaining a biological sample from a subject are known in theart. For example, methods of obtaining lung tissue or lung cells areroutine. For example, a sample from a lung tumor that contains cellularmaterial can be obtained by surgical excision of all or part of thetumor, by collecting a fine needle aspirate from the tumor, as well asother methods known in the art. In some examples, the sample is obtainedfrom a subject having or suspected to have NSCLC. In particularexamples, the sample obtained from the subject includes NSCLC tumorcells, such as at least a portion of an NSCLC tumor. In some examples,the sample from the subject also includes normal (e.g., non-tumor)tissue or cells.

Samples are processed post-collection by fixation and in some examplesare wax- (e.g., paraffin-) embedded. Fixatives for mounted cell andtissue preparations are well known in the art and include, withoutlimitation, formalin fixative, 95% alcoholic Bouin's fixative; 95%alcohol fixative; B5 fixative, Bouin's fixative, Karnovsky's fixative(glutaraldehyde), Hartman's fixative, Hollande's fixative, Orth'ssolution (dichromate fixative), and Zenker's fixative (see, e.g.,Carson, Histotechology: A Self-Instructional Text, Chicago: ASCP Press,1997). ALK staining intensity may decrease if particular fixatives (suchas 95% alcohol, AFA, B5, or Prefer) are used. ALK staining may alsodecrease if tissue samples are not fixed within a short time ofcollection or are not fixed for a sufficient period of time. Inparticular examples, the sample is fixed in neutral buffered formalin(such as 10% neutral buffered formalin) or zinc formalin. In someexamples, the sample is fixed for at least about 6 hours (for example,about 6-48 hours, 12-24 hours or about 6, 12, 16, 18, 24, 36, or 48hours). In additional examples, the sample is placed in fixative withinabout 6 hours of collection (for example, within about 15 minutes, 30minutes, 1, 2, 3, 4, 5, or 6 hours).

In some examples, the sample can be a fixed, wax-embedded lung tissuesample, such as a fixed, wax-embedded lung tissue sample including NSCLCtumor. In some examples, the sample is a lung tissue section includingNSCLC tumor that is stained with hematoxylin and eosin (H&E).

In some examples, the sample is a lung tissue section including NSCLCtumor labeled with a primary antibody specific for ALK, which may belabeled directly or indirectly (e.g., with a labeled secondaryantibody), which in some examples is further stained with H&E.

In some examples, the sample (or a fraction thereof) is present on asolid support. Solid supports bear the biological sample and permit theconvenient detection of components (e.g., proteins) in the sample.Exemplary supports or substrates include microscope slides (e.g., glassmicroscope slides or plastic microscope slides), coverslips (e.g., glasscoverslips or plastic coverslips), tissue culture dishes, multi-wellplates, membranes (e.g., nitrocellulose or polyvinylidene fluoride(PVDF)) or BIACORE™ chips.

D. Methods of Treatment

The disclosed embodiments can further include selecting subjects fortreatment with an ALK inhibitor, for example if the sample from thesubject is scored as ALK-positive. Additionally, the disclosed methodscan further include administering an ALK inhibitor to the subject if thesample from the subject is scored as ALK-positive.

In some examples, the ALK inhibitor is a small molecule inhibitor, suchas crizotinib (Pfizer, New York, N.Y.), AP26113 (Ariad Pharmaceuticals,Cambridge, Mass.), CH5424802 (Chugai Pharmaceutical, Tokyo, Japan),LDK378 (Novartis, Basel, Switzerland), ASP3026 (Astellas Pharma,Northbrook, Ill.), X-396 (Xcovery, West Palm Beach, Fla.), orretaspimycin (Infinity Pharmaceuticals, Cambridge, Mass.). AdditionalALK inhibitors include 3-39 (Novartis), GSK1838705A (GlaxoSmithKline,Boston, Mass.), and CEP-28122 (Cephalon, Frazer, Pa.). In anotherexample, an ALK inhibitor is an anti-ALK antibody, such as a humanizedanti-ALK antibody.

In some examples, the disclosed methods include providing atherapeutically effective amount of the ALK inhibitor to the subject(such as a subject having ALK-positive NSCLC). Methods and therapeuticdosages of such agents and treatments are known to those of ordinaryskill in the art, and for example, can be determined by a skilledclinician. In a non-limiting example, a therapeutically effective amountof crizotinib is administered to a subject having an NSCLC tumor that isidentified as ALK-positive. In some examples, a therapeuticallyeffective amount of crizotinib can be about 50-2000 mg/day (such asabout 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 mg/day),administered orally in one or two doses per day. In some examples, themethods include orally administering 200 mg of crizotinib to the subjectonce or twice per day if the sample from the subject is scored asALK-positive. In other examples, the methods include orallyadministering 250 mg of crizotinib to the subject once or twice per dayif the sample from the subject is scored as ALK-positive. Dosages anddosing schedules of crizotinib for a subject can be determined by askilled clinician, taking into account additional factors such as tumorsite, tumor stage, tumor grade, patient treatment history, patientperformance and nutritional status, concomitant health problems, socialand logistic factors, previous primary tumors, and patient preference.Crizotinib may be administered on a continuous dosing schedule oradministered for one or more cycles (for example, one or more cycles of21-28 days). Treatment may repeat every 21-28 days if administered incycles.

In some examples, the subject is also administered one or moreadditional chemotherapeutic agents in combination with the ALKinhibitor. Additional chemotherapeutic agents include, but are notlimited to alkylating agents, such as nitrogen mustards (for example,chlorambucil, chlormethine, cyclophosphamide, ifosfamide, andmelphalan), nitrosoureas (for example, carmustine, fotemustine,lomustine, and streptozocin), platinum compounds (for example,carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan,dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, anduramustine; antimetabolites, such as folic acid (for example,methotrexate, pemetrexed, and raltitrexed), purine (for example,cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine),pyrimidine (for example, capecitabine), cytarabine, fluorouracil, andgemcitabine; plant alkaloids, such as podophyllum (for example,etoposide, and teniposide), taxane (for example, docetaxel andpaclitaxel), vinca (for example, vinblastine, vincristine, vindesine,and vinorelbine); cytotoxic/antitumor antibiotics, such as anthracyclinefamily members (for example, daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin,hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecanand irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab,cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, andtrastuzumab; photosensitizers, such as aminolevulinic acid, methylaminolevulinate, porfimer sodium, and verteporfin; and other agents,such as alitretinoin, altretamine, amsacrine, anagrelide, arsenictrioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib,celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib,hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin,masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib,vemurafinib, vandetanib, and tretinoin. Selection and therapeuticdosages of such agents are known to those skilled in the art, and can bedetermined by a skilled clinician.

In other examples, the disclosed methods include providing surgery,radiation therapy, and/or chemotherapeutics to the subject incombination with the ALK inhibitor (for example, sequentially,substantially simultaneously, or simultaneously). Methods andtherapeutic dosages of such agents and treatments are known to thoseskilled in the art, and can be determined by a skilled clinician.

The present disclosure is illustrated by the following non-limitingExamples.

Example 1 ALK Immunohistochemistry Protocol

This example describes methods of ALK IHC for identifying ALK-positiveNSCLC samples.

Formalin-fixed paraffin embedded NSCLC tissue sections were stained withanti-ALK D5F3 rabbit monoclonal antibody (Ventana Medical Systems,Catalog No. 790-4794). A separate tissue section from each NSCLC samplewas stained with Rabbit Monoclonal Negative Control Ig (Ventana, CatalogNo. 790-4795). Slides were also stained with hematoxylin and eosin (H&E)to distinguish morphology. ALK 2 in 1 control slides (Ventana CatalogNo. 781-4796) were used as system level positive and negative controls.Slides were stained using a Ventana BENCHMARK XT automated slide stainerand OPTIVIEW DAB IHC Detection Kit (Ventana, Catalog No. 760-700) andOPTIVIEW Amplification Kit (Ventana Catalog No. 760-099) with thestaining protocol shown in Table 1. The same protocol and reagents werealso tested using a Ventana BENCHMARK GX automated slide stainer.

TABLE 1 Staining Protocol for anti-ALK (D5F3) and Rabbit MonoclonalNegative Control Ig with OPTIVEIW DAB IHC Detection Kit and OPTIVIEWAmplification Kit Procedure Type Method Deparaffinization Selected CellConditioning (antigen unmasking) Cell Conditioning 1 92 minutes, 100° C.Pre-Primary Peroxidase Inhibitor Selected Antibody (Primary) 16 minutes,37° C. OPTIVIEW HQ Univ Linker 12 minutes OPTIVIEW HRP Multimer 12minutes OPTIVIEW Amplification Selected OV AMP H2O2, OV Amplifier 8minutes OV AMP Multimer 8 minutes Counterstain Hematoxylin II, 4 minutesPost Counterstain Bluing, 4 minutes

Example 2 Evaluation of Immunohistochemical Results

This example describes exemplary methods of evaluating or scoring ALKIHC results to determine whether an NSCLC sample is positive or negativefor ALK.

ALK IHC was performed with the anti-ALK D5F3 antibody as described inExample 1. Neoplastic cells labeled with the ALK IHC assay wereevaluated for presence or absence of DAB signal. The matched negativecontrol slide was used to assess non-specific background staining anddegree of background staining known to occur due to specific tissueelements (alveolar macrophages, neural cells, glandular epithelialcells, cells in lymphocytic infiltrate). Samples were determined to bepositive or negative for ALK based on the scoring algorithm in Table 2.

TABLE 2 Scoring criteria for determination of ALK status in NSCLCClinical Interpretation Staining Description Positive Presence of stronggranular cytoplasmic staining in tumor for ALK cells (any percentage ofpositive tumor cells). Known staining elements should be excluded,including: light cytoplasmic stippling in alveolar macrophages cells ofneural origin (nerve and ganglion cells) glandular epithelial stainingcells within lymphocytic infiltrate Some background staining may also beobserved within normal mucosa in NSCLC (including mucin) and in necrotictumor areas, which should also be excluded from the clinical evaluationNegative Absence of strong granular cytoplasmic staining in tumor forALK cells

Positive for ALK:

Positive cases stained with the IHC assay described in Example 1typically displayed a strong, granular cytoplasmic signal. Any samplethat had strong cytoplasmic staining (such as 3+ staining inconventional IHC scoring) in any number of tumor cells was scored aspositive. In the majority of positive cases, the signal was distributedhomogeneously, having a uniform level of intensity throughout theneoplastic portions of the tumor. In some positive cases, the signal wasmore heterogeneous in staining intensity. Examples of homogeneous ALKIHC expression are shown in FIGS. 1A and B. Examples of heterogeneousALK IHC expression are shown in FIGS. 2A-C.

Some background staining was observed within normal mucosa in NSCLCsamples, as well as in necrotic tumor areas. This staining was notevaluated as ALK-positive staining. Additionally, staining was noted inneural cells (including nerve or ganglion cells), glandular epithelialcells, alveolar macrophages, and cells in lymphocytic infiltration orlung metastases to lymph nodes. Staining of mucin was also noted in somesamples. All of these were excluded when determining whether the samplewas ALK-positive. Examples of these excluded types of ALK staining areshown in FIGS. 3 (glandular epithelial cell staining), 4A and B (neuralcell staining), 5 (alveolar macrophage staining), 6A and B (mucinstaining), and 7A and B (lymphocyte staining).

Negative for ALK:

The majority of ALK-negative cases exhibited an absence of DAB signalabove background staining from the matched negative control slide (FIGS.8A and B). However, a minority of negative cases displayed a weak,diffuse granular cytoplasmic pattern that was detected above backgroundstaining observed on the matched negative control slide (FIGS. 9A andB). These cases were estimated to represent about 1-2% of ALK-negativecases and were negative by confirmatory FISH analysis.

In a few cases, cytoplasmic staining on the slide stained with theanti-ALK (D5F3) antibody was more notable than on the negative controlslide. However, these cases did not exhibit strong cytoplasmic staining,and were therefore determined to be ALK-negative (FIG. 10A-D). Inanother case, membrane/cytoplasmic staining was observed in a slidestained with the anti-ALK antibody (FIG. 11). This sample was determinedto be ALK-negative due to the lack of strong granular cytoplasmicstaining.

Example 3 Concordance of Immunohistochemical Results with FISH

This example demonstrates the concordance of the ALK IHC scoring methoddescribed in Example 2 with ALK break-apart FISH for determining ALKstatus of NSCLC samples.

Three cohorts were used to compare the staining results from theanti-ALK (D5F3) Rabbit Monoclonal Primary Antibody with ALK FISH interms of ALK clinical status. The cohorts included a range of humanNSCLC tissue samples from primary and metastatic tumors, includingresections, needle biopsies, bronchial biopsies, and formalin-fixed,paraffin-embedded (FFPE) cell blocks from fine needle aspirates. IHC wascarried out as described in Example 1. All studies were scored using thescoring algorithm in Example 2 (described in Table 2). Break apart FISHdata was obtained using the VYSIS ALK Break Apart FISH Probe Kit (AbbottLaboratories, Abbott Park, Ill.) according to the manufacturer'sprotocols.

Concordance Study 1

A study was conducted in an external laboratory comparing the anti-ALK(D5F3) Rabbit Monoclonal Primary Antibody with retrospective ALK breakapart FISH data (Cleveland Clinic Foundation). The external site stainedabout 100 NSCLC cases using the anti-ALK (D5F3) Rabbit MonoclonalPrimary Antibody on a BenchMark XT instrument. The anti-ALK IHC assaydemonstrated >98% overall percent agreement with the retrospective ALKbreak apart FISH data on this NSCLC sample cohort. The results aredetailed in Tables 3 and 4. Of the 100 cases, 86 had available FISH dataand sufficient tumor present for comparison with the ALK IHC result. Thepreparation of the tissue specimens for this study was not verified asto fixation conditions (time to fixation after collection, time offixation).

TABLE 3 Anti-ALK D5F3 IHC compared to break apart FISH Break Apart FISHAnti-ALK D5F3 Positive Negative Total Positive 10 0 10 Negative 1 75 76Total 11 75 86

TABLE 4 Percent overall, positive, and negative agreement rates foranti-ALK D5F3 IHC compared to break apart FISH Rate n/N % 95% CI^(a)Overall Percent Agreement 85/86 98.8 93.7, 99.8 Positive PercentAgreement 10/11 90.9 62.3, 98.4 Negative Percent Agreement 75/75 100.095.1, 100.01 ^(a)Two-sided 95% confidence interval calculated using thescore method

Concordance Study 2

This study was conducted in a second external laboratory comparing theanti-ALK (D5F3) Rabbit Monoclonal Primary Antibody with ALK break apartFISH data on 73 NSCLC cases (cut within one week of staining). Theexternal site stained the cases using the anti-ALK (D5F3) RabbitMonoclonal Primary Antibody on a BenchMark XT instrument as described inExample 1. The VENTANA ALK IHC assay demonstrated >93% overall percentagreement with the retrospective ALK break apart FISH data on this NSCLCsample cohort. The results are detailed in Tables 5 and 6.

TABLE 5 Anti-ALK D5F3 IHC compared to break apart FISH Break Apart FISHAnti-ALK D5F3 Positive Negative Total Positive 2 4 6 Negative 0 56 56Total 2 60 62

TABLE 6 Percent overall, positive, and negative agreement rates foranti-ALK D5F3 IHC compared to break apart FISH Rate n/N % 95% CI^(a)Overall Percent Agreement 58/62 93.5 84.6-97.5 Positive PercentAgreement 2/2 100 34.2-100.0 Negative Percent Agreement 56/60 9384.1-97.4 ^(a)Two-sided 95% confidence interval calculated using thescore method

Of the four discordant (FISH negative, ALK IHC positive) cases,additional unstained slides were tested with another ALK IHC assay(different clone and detection system). Three of the four cases agreedwith the anti-ALK (D5F3) IHC assay in terms of ALK IHC stainingdetected.

There were also 10 cases where FISH results were undetermined or was notperformed. Four of these cases were positive by the anti-ALK (D5F3) IHCassay and the other ALK clone, and six were negative by ALK IHC. Therewas one case that was positive by FISH but not enough sample wasavailable to stain with IHC.

Concordance Study 3

In this study, about 300 cases from an on-going, global clinical studyof ALK positive NSCLC patients enrolled with the ALK break apart FISHProbe study were stained with the anti-ALK (D5F3) Rabbit MonoclonalAntibody assay. Of the about 300 cases, some were categorized as“uninformative” by FISH or “FISH assay not performed” and were stainedand evaluated for informational purposes only. FISH data for the sampleswere obtained from central labs participating in the clinical study.

The cases were blinded for FISH status, randomized, and provided to tworeaders, who evaluated the staining results. Results were compared withthe FISH status obtained from the global clinical study. Results of thecomparison of ALK IHC with ALK break apart FISH are shown in Table 7.The preparation of the tissue specimens for this study was not verifiedas to fixation conditions (time to fixation after collection, time offixation).

TABLE 7 Agreement of anti-ALK D5F3 IHC with ALK break apart FISH asevaluated by two pathologists. Anti-ALK D5F3 ALK Break Apart FISH ReaderPositive Negative Total Reader 1 Positive 37 13 50 Negative 11 223 234Total 48 236 284 Reader 2 Positive 37 12 49 Negative 11 225 236 Total 48237 285 95% Confidence Reader n/N Percent Interval Reader 1 OverallAgreement 260/284 91.5 87.7, 94.3 Positive Agreement 37/48 77.1 63.5,86.7 Negative Agreement 223/236 94.5 90.8, 96.8 Reader 2 OverallAgreement 262/285 91.9 88.2, 94.6 Positive Agreement 37/48 77.1 63.5,86.7 Negative Agreement 225/237 94.9 91.4, 97.1

Discrepant cases that were ALK IHC positive, ALK FISH negative:

-   -   Four cases were evaluated by at least one reader as ALK IHC        positive, FISH negative. Upon consensus review, it was        determined that they should be evaluated as IHC negative. These        cases had focal cytoplasmic/membrane staining and are considered        to be negative, as described in Example 2.    -   There were nine ALK IHC positive, ALK FISH negative cases that        were considered true discrepant cases.

Of the nine discordant cases, seven had unstained slides that wereavailable for additional ALK diagnostic testing (molecular testing andIHC testing using a different clone and detection system). Theseadditional testing results indicated that the majority of discrepantcases favored the positive IHC evaluation for ALK status when ALK FISHwas negative. The slides from these cases were cut more than threemonths prior to staining, which may decrease sensitivity of the IHCassay.

Discrepant cases that were ALK IHC negative, ALK FISH positive:

-   -   There were 11 cases that were positive by FISH but negative by        the anti-ALK IHC assay. Ten of these cases had unstained slides        that were available for the additional ALK diagnostic testing        with molecular techniques and IHC. These additional testing        results indicated that the majority of cases that were negative        by the anti-ALK D5F3 IHC assay were also negative by another ALK        IHC system, but were positive by one or more molecular assays.        The slides from these cases were cut more than three months        prior to staining, which may decrease sensitivity of the IHC        assay.    -   There were 14 cases in this cohort that were uninformative by        FISH (no result was obtained). Of these, three were evaluated as        positive by both readers by the anti-ALK IHC assay. In addition,        there were 19 cases where the FISH assay could not be performed,        based on the H&E slide (usually due to the tumor content being        insufficient). Of these, both readers evaluated the ALK IHC        staining results as positive in four cases. Therefore, on        average, 21% of the cases where FISH results were not obtained        had a positive ALK status by the anti-ALK (D5F3) IHC assay.

The reproducibility of the ALK status as obtained by the anti-ALK IHCassay was determined. The ALK status obtained by each reader for theabout 300 cases was compared. The results indicate that the anti-ALK IHCassay and scoring algorithm were highly reproducible between readers, asshown in Table 8.

TABLE 8 Scoring interpretation inter-reader precision Reader ComparisonALK Break Apart FISH Reader 2 vs. Reader 1 Positive Negative TotalAnti-ALK Positive 56 0 56 D5F3 IHC Negative 1 251 252 Total 57 251 30895% Confidence Reader Comparison n/N Percent Interval Reader 2 vs.Overall Percent 307/308 99.7 98.2, 99.9  Reader 1 Agreement AveragePositive 112/113 99.1 97.0, 100.0 Agreement Average Negative 502/50399.8 99.4, 100.0 Agreement

CONCLUSION

IHC with the anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody wasreproducible in its staining results for clinical ALK status on theBENCHMARK XT and BENCHMARK GX platforms. The binary scoring algorithmwas highly reproducible across readers. The assay was concordant withthe ALK break apart FISH method for ALK status.

Example 4 Effect of Tissue Processing Conditions on IHC Assay

This example describes the effect of varying tissue processingconditions on the IHC assay.

The H228 (ALK-positive) cell line was used to generate xenograft tumorsin SCID mice. The tumors were harvested and fixed with differentfixatives for varying times and were stained with the anti-ALK D5F3antibody as described in Example 1.

Tissues fixed with 10% neutral buffered formalin for at least 6 hoursyielded optimal ALK IHC staining results. Zinc formalin fixation for atleast 6 hours also yielded acceptable ALK IHC staining. Fixation time ofless than 6 hours in either neutral buffered formalin or zinc formalinresulted in significantly decreased staining intensity for ALK.Additional fixatives were also tested (AFA, B5, Prefer, and 95% ethanol)and resulted in significantly decreased ALK staining intensity at alltime points.

Xenograft samples were harvested and left unfixed for times ranging from30 minutes to 24 hours, then fixed for 12 hours in neutral bufferedformalin. ALK staining intensity was decreased if the time to fixationwas delayed more than 6 hours.

Sections that had been cut, but not stained, for various periods of timewere also assessed for ALK IHC staining. ALK staining intensitydecreased in sections that had been cut more than 3 months beforestaining and stored at room temperature. Despite decreased stainingintensity, none of the ALK-positive cases changed status toALK-negative.

Based on these data, fixation of samples within about 6 hours ofcollection in 10% neutral buffered formalin or zinc formalin for atleast about 6 hours is recommended for optimal ALK staining results. Inaddition, staining is optimal if sections are stained within about 3months of cutting, when stored at room temperature.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

1. A method of identifying a subject as having non-small cell lungcarcinoma (NSCLC) likely to respond to treatment with an anaplasticlymphoma kinase (ALK) inhibitor, comprising: contacting a samplecomprising NSCLC tumor cells from the subject with an anti-ALK antibody;detecting ALK protein expression in the sample; scoring the sample aspositive or negative for ALK; and identifying the subject as havingNSCLC likely to respond to treatment with an ALK inhibitor if the sampleis scored as positive for ALK or identifying the subject as having NSCLCnot likely to respond to treatment with an ALK inhibitor if the sampleis scored as negative for ALK.
 2. The method of claim 1, wherein scoringthe sample as positive or negative for ALK comprises: scoring the sampleas positive for ALK if detecting the ALK protein expression comprisesdetecting strong granular cytoplasmic staining in one or more tumorcells in the sample; or scoring the sample as negative for ALK ifdetecting the ALK protein expression does not comprise detecting stronggranular cytoplasmic staining in one or more tumor cells in the sample.3. The method of claim 2, wherein scoring the sample as positive ornegative for ALK further comprises excluding detecting ALK proteinexpression in normal cells or elements or necrotic tumor areas in thesample.
 4. The method of claim 3, wherein the normal cells in the samplecomprise non-tumor cells, cells of neural origin, alveolar macrophages,glandular epithelial cells, or lymphocytes.
 5. The method of claim 3,wherein the normal elements comprise mucin.
 6. The method of claim 1,wherein the sample comprises a tissue biopsy, fine needle aspirate,bronchoalveolar lavage, pleural fluid, or sputum.
 7. The method of claim6, wherein the tissue biopsy comprises a tissue section.
 8. The methodof claim 6, wherein the sample is fixed.
 9. The method of claim 8,wherein the sample is fixed for at least about 6 hours inneutral-buffered formalin or zinc formalin within about 6 hours of thesample collection.
 10. The method of claim 1, wherein the anti-ALKantibody is a rabbit anti-ALK D5F3 antibody.
 11. The method of claim 1,wherein detecting ALK protein expression in the sample comprises director indirect detection of binding of the anti-ALK antibody to the sample.12. The method of claim 1, further comprising a signal amplificationstep.
 13. The method of claim 1, wherein contacting the sample with ananti-ALK antibody and detecting the ALK protein expression in the sampleare performed with an automated tissue stainer.
 14. The method of claim2, wherein scoring the sample as positive or negative for ALK comprisesvisual inspection.
 15. The method of claim 14, wherein the visualinspection is performed utilizing light microscopy.
 16. The method ofclaim 14, wherein scoring the sample as positive or negative for ALKcomprises visual inspection of at least one field of view.
 17. Themethod of claim 1, further comprising selecting the subject fortreatment with an ALK inhibitor if the sample from the subject is scoredas positive for ALK.
 18. The method of claim 17, further comprisingadministering a therapeutically effective amount of the ALK inhibitor tothe selected subject.
 19. The method of claim 1, wherein the ALKinhibitor comprises crizotinib.
 20. A method of identifying a subject ashaving non-small cell lung carcinoma (NSCLC) likely to respond totreatment with crizotinib, comprising: (a) contacting a samplecomprising NSCLC tumor cells from the subject with an rabbit anti-ALKD5F3 antibody; (b) detecting ALK protein expression in the sample,comprising: (i) contacting the sample with a3-hydroxyquinoxaline-2-carboxylic acid-conjugated secondary antibodywhich binds to the anti-ALK antibody; (ii) contacting the sample with ahorseradish peroxidase-conjugated tertiary antibody which binds to the3-hydroxyquinoxaline-2-carboxylic acid; (iii) contacting the sample withhydrogen peroxide and 3,3′-diaminobenzidine tetrahydrochloride; and (iv)detecting the DAB precipitate utilizing light microscopy; (c) scoringthe sample as positive or negative for ALK, comprising: (i) scoring thesample as positive for ALK if strong granular cytoplasmic staining ispresent in one or more tumor cells in the sample; or (ii) scoring thesample as negative for ALK if strong granular cytoplasmic staining isnot present in one or more tumor cells in the sample; and (d)identifying the subject as having NSCLC likely to respond to treatmentwith crizotinib if the sample is scored as positive for ALK oridentifying the subject as having NSCLC not likely to respond totreatment with crizotinib if the sample is scored as negative for ALK.21. The method of claim 20, wherein detecting ALK protein expression inthe sample further comprises after step (b)(ii): contacting the samplewith hydrogen peroxide and tyramide conjugated3-hydroxyquinoxaline-2-carboxylic acid under conditions sufficient fordeposition of the 3-hydroxyquinoxaline-2-carboxylic acid at or near theALK protein; and contacting the sample with the tertiary antibody whichbinds to the 3-hydroxyquinoxaline-2-carboxylic acid.
 22. The method ofclaim 21, further comprising selecting the subject for treatment withcrizotinib if the sample from the subject is scored as positive for ALK.23. The method of claim 22, further comprising administering atherapeutically effective amount of crizotinib to the selected subject.